Identification of ses-1 and the uses of the same

ABSTRACT

The invention relates to isolated nucleic acid molecules coding for SES-1 proteins or muted SES-1 proteins, and vectors and transgenic organisms containing such nucleic acid molecules. The invention also relates to uses of such nucleic acid molecules for producing pharmaceuticals and for producing model organisms. The invention further relates to the corresponding SES-1 proteins and muted SES-1 proteins, and the antibodies induced thereby. Finally, the invention relates to the use of substances which increase the expression of human presenilin, for the treatment of Alzheimer&#39;s disease, in addition to said substances themselves and pharmaceutical compositions containing the same.

The present invention relates to isolated nucleic acid molecules whichencode ses-1 or mutated ses-1, to vectors and transgenic organisms whichcontain these nucleic acid molecules, to uses of these nucleic acidmolecules for producing pharmaceuticals and for generating modelorganisms, to the use of transgenic organisms in methods for identifyingsubstances which alter ses-1 activity or, respectively, increasepresenilin activity (e.g. that of sel-12 or hop-1), and to thecorresponding SES-1 proteins and mutated SES-1 proteins as well asantibodies to which these proteins give rise. The invention furthermorerelates to the use of substances which increase the expression of ahuman presenilin for treating Alzheimer's disease, and to thesesubstances themselves and to pharmaceutical compositions which comprisethese substances.

BACKGROUND OF THE INVENTION

Aside from Parkinson's disease, Alzheimer's disease is thatneuro-degenerative disease which is most well known. The characteristicfeature of Alzheimer's disease is the development of neuronal proteinaggregations, what are termed plaques, which are essentially composed ofan insoluble peptide, of 4 kDa in size, termed amyloid beta-peptide(Aβ4). Investigations carried out in the last few years indicate thatthe formation of Aβ4 is causatively involved in the development of thedisease. Dominant mutations in three genes give rise to familial formsof the disease (FAD, familial Alzheimer's disease). It has been foundthat one of the genes concerned encodes amyloid precursor protein (APP),which can be processed by three proteases, resulting in the formation ofAβ4, inter alia. FAD mutations in APP increase the quantity of Aβ42, a42 amino acid-long variant of Aβ4, which is produced.

Molecular genetic investigations of families in which Alzheimer'sdisease has occurred have led to the identification of the human genespresenilin 1 and presenilin 2 (Rogaev et al. 1995, Nature 376, 775-778;Levy-Lahad, E. et al. 1995, Science 269: 973-977) which, when altered byparticular mutations, are causatively involved in the onset ofAlzheimer's disease and which also play a key role in the Notch signaltransduction pathway during development of the disease. Presenilinproteins are located in the ER-Golgi and possess at least 6transmembrane domains. It has been found that mutations in PS1 and PS2influence the formation of Aβ4 and are involved in giving rise toAlzheimer's disease, presumably by means of the formation of amyloiddeposits. Members of the presenilin family have been identified in themouse, in Drosophila melanogaster, in Xenopus laevis and in thethreadworm Caenorhabditis elegans, inter alia. Although mutatedpresenilins influence the processing of APP in humans, their naturalfunction in humans is not known in detail; it has been postulated thatthey might function as an APP protease (gamma-secretase). Otherinvestigations carried out on human cell cultures have indicated thatpresenilins play a role in the intracellular proteolysis of Notch-likereceptors after they have been activated by ligand binding. It isassumed, therefore, that the presenilins are involved in processing atleast two different membrane proteins (APP and Notch). At present, it isa controversial matter as to whether the presenilins themselves are theproteases in this processing or whether they are cofactors of thesereactions or influence the proteolysis indirectly (Haass, C. et al.1999, Science 286 (5441): 916-919).

Taking the abovementioned presenilin genes as the starting point,intensive research is being carried out to elucidate the moleculardisease processes and to develop pharmaceuticals for treating andpreventing Alzheimer's disease. Factors which influence the activity ofthe presenilin genes are being sought, in particular, particularimportance being attached, in this connection, to the search forsuppressors of the presenilin mutant phenotype. Suppressors can bemutations in other genes which eliminate the requirement for thepresenilin function or they can be substances which modulate theactivity of other genes, or of the presenilins, such that the presenilinmutant defect does not have any phenotypic consequences.

A nematode, in particular Caenorhabditis elegans, has frequently beenemployed as a preferred model organism in these investigations. Theadvantage of a nematode model, in particular of the C. elegans model,lies, in particular, in its suitability for a high-throughput method(HTS; high-throughput screening), the possibility of faster geneticanalysis due to a shorter generation time (2-3 days) and detailedknowledge of the molecular and functional properties of the nervoussystem in C. elegans. Since C. elegans can be kept on microtiter plates,it is possible to use this test system to test, by means of HTS, 10 000or more substances on the living worm over a short period of time.

Three presenilin genes, designated sel-12, hop-1 and spe-4, have thusfar been identified in the threadworm C. elegans. Spe-4 is the mostdivergent member of the family. Mutations in spe-4 lead to a defect incytoplasmic partitioning during spermatogenesis (L'Hemault, S. W. et al.1992, J Cell Biol 119: 55-68). While mutations in hop-1 do not have anyvisible phenotype, they lead, in combination with mutations in sel-12,to a synthetically lethal phenotype (Li, X. et al. 1997, Proc Natl AcadSci USA 94: 12204-12209). Sel-12 is the gene which most stronglyresembles human PS1 and PS2 and is also the best-studied presenilin genein C. elegans. It has been found that certain sel-12 mutants exhibit anegg-laying defect and also morphological changes in vulva development(Levitan, D. et al. 1995, Nature 377: 35-14). Sel-12 is 50% identicalwith human presenilins, and the egg-laying defect in sel-12 mutants canbe offset by transgenically expressing PS1 or PS2 from the sel-12promoter (Baumeister et al., 1997, Genes and Function 1: 149-159). Thisdemonstrates that PS1 and PS2 exhibit functional homology with sel-12.Sel-12 mutations were initially identified as suppressors of themultivulva phenotype of a gain-of-function mutant in the lin-12 andglp-1 Notch receptor gene. Notch receptors control determination of cellfate in many multicellular organisms. In addition, it has been shownthat the phenotype of sel-12 mutants resembles that of weakfunction-loss mutants of lin-12. In summary, it can be stated thatpresenilins play an important role in Notch signal transduction in alarge number of organisms, including mammals.

SUMMARY OF THE INVENTION

The present inventors have now found, surprisingly, that certainmutations, as shown in FIG. 1 for example, in another gene, i.e. the C.elegans ses-1 gene, which was first identified by the inventors, areable to suppress a defect, in particular the egg-laying defect of asel-12 mutant. This means that particular mutations in the ses-1 genelead, for example, to the egg-laying defect, which is caused by certainsel-12 mutations, being eliminated and the double mutants (sel-12⁻;ses-1⁻) once again exhibiting a normal wild-type phenotype. Moredetailed investigations have shown that these mutations in the ses-1gene lead, in the sel-12 mutants, to hop-1 being activated, i.e. thehop-1 presenilin protein takes over the function of the defective sel-12presenilin protein.

The present inventors have located the novel ses-1 gene on the C.elegans genome and sequenced it.

Consequently, the present invention initially relates to an isolatednucleic acid molecule which encodes SES-1 protein and to other nucleicacid molecules which encode mutated SES-1 proteins.

In addition to this, the invention relates to a vector which comprisesthese isolated nucleic acid molecules, or fragments thereof.

The invention furthermore relates to the SES-1 proteins and mutatedSES-1 proteins, or fragments thereof, which are encoded by the nucleicacid molecules and to antibodies which are generated using theseproteins.

In addition, the invention relates to a transgenic, nonhuman organismwhich comprises an isolated nucleic acid molecule which encodes SES-1protein or mutated SES-1 protein. In particular, the invention relatesto transgenic C. elegans organisms which exhibit an ses-1 allele whichdecreases the activity of ses-1 and, respectively, increases theactivity of sel-12 or hop-1. In this connection, transgenic organismsare understood as being those organisms in which the genome has beenaltered by mutation, and which exhibit a mutated ses-1 gene as a result,or from which the ses-1 gene has been removed.

Finally, the invention relates to uses of these nucleic acid moleculesfor producing a pharmaceutical directed against Alzheimer's disease andfor generating model systems for investigating this disease stillfurther.

In particular, the invention relates to the use of transgenic C. elegansin a method for identifying and/or characterizing substances whichreduce the activity of ses-1 or, respectively, increase the activity ofsel-12 or hop-1.

The invention furthermore also relates to the use of substances whichincrease the expression of human presenilin 1 or 2 for treatingAlzheimer's disease and to these substances themselves and topharmaceutical compositions which comprise these substances.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the fact that it has been possible toidentify a novel gene, ses-1, in C. elegans, with this gene interactingwith the C. elegans presenilin genes sel-12 and hop-1 and therebyplaying an important role in the development of Alzheimer's disease.

Thus, various mutations in the ses-1 gene, including complete deletionof the ses-1 gene as well, resulted in certain altered phenotypes whichwere elicited by sel-12 mutations being suppressed. The suppression ofthese sel-12 defects in ses-1 mutants is achieved by the latter markedlyderepressing-expression of hop-1 and by the HOP-1 protein presumablybeing able to take over the function of the defective sel-12 protein.

The ses-1 gene which has been identified contained up to 7 predictedC2H2 zinc finger domains and two regions which could serve as nuclearlocalization signals (FIG. 4).

When the BLAST program was used to compare its sequence with the Genbankdatabase, the deduced SES-1 protein did not show any clear similarity toany other known protein. All that was observed was a slight similarityto a variety of proteins which also contains zinc finger domains.

Consequently, the present invention provides an isolated nucleic acidmolecule which encodes SES-1 protein.

The invention furthermore encompasses isolated nucleic acid moleculeswhich encode mutated SES-1 proteins. In particular, the inventionprovides isolated nucleic acid molecules which encode mutated SES-1proteins which have at least one of the mutations given in FIG. 1.

In addition to this, other aspects of the invention relate to isolatednucleic acid molecules, in particular DNA, such as cDNA or genomic DNA,molecules, but also RNA molecules, for example, which encode a C.elegans SES-1 protein having the amino acid sequence given in SEQ ID NO:1 or SEQ ID NO: 2, in particular those which exhibit a nucleic acidsequence given in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ IDNO: 6.

The invention also encompasses isolated nucleic acid molecules whosenucleotide sequences exhibit at least 75%, in particular at least 80%,especially at least 85%, preferably at least 90%, particularlypreferably at least 95% and most preferably at least 98%, sequencesimilarity to the abovementioned nucleic acid molecules, in particularthose which hybridize with the abovementioned nucleic acid moleculesunder stringent conditions. Such a hybridization preferably takes placeunder conditions of low stringency; in another embodiment, it also takesplace under conditions of high stringency. In the context of thisdescription, conditions of low stringency are understood as meaning ahybridization in 3×SSC at from room temperature to 65° C. and conditionsof high stringency are understood as meaning a hybridization in 0.1×SSCat 68° C. SSC is the abbreviation for a 0.15 M sodium chloride, 0.015 Mtrisodium citrate buffer.

The nucleic acid molecules according to the invention having nucleicacid sequence similarity to the nucleic acid sequence given in SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 also comprise, inparticular, allelic variants of the given nucleic acid sequence, whichvariants include, in particular, the abovementioned mutations.

The invention also extends to the nucleic acid molecules which in eachcase have a nucleic acid sequence which is complementary to the nucleicacid sequences described above.

In the present context, the term “isolated nucleic acid molecule” refersto nucleic acid molecules which are present in a form in which they areessentially purified from the main quantity of nucleic acid molecules ofdiffering nature derived from the starting cells or producing cells.However, preparations of isolated nucleic acid molecules according tothe invention can perfectly well contain other constituents such assalts, substances from the medium or residual constituents of theproducing cells, such as various proteins.

The invention also encompasses vectors which contain a nucleic acidmolecule according to the invention which encodes SES-1 protein ormutated SES-1 proteins. The invention also includes vectors whichcontain a fragment of a nucleic acid molecule according to theinvention. In this connection, the nucleic acid molecule is linked to apromoter which causes the nucleic acid molecule to be expressed in theorganism which is used for the expression. In this connection, one ofthe known C. elegans promoters(http://chinook.uoregon.edu/promoters.html) is used, in particular, forexpressing in C. elegans.

The vector can, for example, be a plasmid, a cosmid, a bacmid, a virusor a bacteriophage.

Another aspect relates to the polypeptides or proteins which are encodedby the described nucleic acid molecules, in particular C. elegans SES-1protein having the amino acid sequence given in SEQ ID NO: 1 or SEQ IDNO: 2 and the abovementioned mutated SES-1 proteins and alsopolypeptides or proteins which are derived therefrom by means ofsubstitution, modification, deletion, insertion or addition of aminoacids and which exhibit at least 75%, in particular at least 80%,especially at least 85%, preferably at least 90%, particularlypreferably at least 95% and most preferably at least 98% sequencesimilarity to the abovementioned amino acid sequences. The inventionalso encompasses fusion proteins which contain the polypeptides,proteins or protein fragments according to the invention fused to aprotein of a different nature or to a protein segment of a differentnature, e.g. a marker protein or indicator protein. The invention alsoincludes fragments of the abovementioned proteins or polypeptides.

The antibodies which can be obtained using these proteins, and which canbe monoclonal or polyclonal, are likewise encompassed by the invention.

The invention also extends to transgenic organisms, in particularmicroorganisms, e.g. bacteria, viruses, protozoa, fungi and yeasts,algae, plants or animals, and also parts, e.g. cells, and propagationmaterials, e.g. seed, from these transgenic organisms, which comprise arecombinant nucleic acid sequence, where appropriate integrated into achromosome or else extrachromosomally, which contains, as a transgene, anucleic acid molecule according to the invention which encodes a SES-1protein or mutated SES-1 protein, or fragments thereof. Under apreferred aspect, the transgenic organisms will also express thepolypeptide or protein encoded by the abovementioned transgene, i.e. C.elegans SES-1 protein or a polypeptide or protein which is derivedtherefrom.

According to one aspect of the invention, transgenic C. elegansorganisms exhibit an ses-1 allele which decreases, amplifies oreliminates the activity of ses-1. The invention also includes thosetransgenic C. elegans organisms from which the ses-1 gene has beencompletely removed. In particular, the present invention relates totransgenic C. elegans organisms which exhibit an ses-1 allele whichoffsets the egg-laying defect phenotype (Egl) in sel-12 mutants. Inaddition, the present invention relates to transgenic C. elegansorganisms which exhibit an ses-1 allele which increases the activity ofhop-1 or sel-12.

The invention also encompasses the use of the nucleic acid moleculeaccording to the invention for producing a pharmaceutical for treatingneurological, in particular neurodegenerative, diseases, particularlypreferably Alzheimer's disease, or for generating a model organism forthe further investigation and elucidation of neurological, in particularneurodegenerative, diseases.

The abovementioned pharmaceuticals can be obtained, for example, byemploying a nucleic acid molecule according to the invention itself as agene therapy agent or by using such a nucleic acid molecule to constructa model organism and formulating the substances which are found usingthis model organism into a pharmaceutical. For further details in thisregard, the reader is referred, for example, to internationalapplication PCT/EP01/03214 belonging to the same applicant.

In particular, the invention relates to the use of transgenic C. elegansorganisms, which contain a nucleic acid molecule according to theinvention, in a method for identifying and/or characterizing substanceswhich alter, in particular decrease or eliminate, the activity of ses-1.The invention furthermore relates to the use of transgenic C. elegansorganisms, which contain a nucleic acid molecule according to theinvention, in a method for identifying and/or characterizing substanceswhich increase the activity of hop-1 or sel-12.

The invention furthermore relates to the use of these same transgenic C.elegans organisms in a method for identifying and/or characterizingsubstances which can be used as active compounds for treating and/orpreventing Alzheimer's disease. In view of the analogy with human cells,and against the background of the demonstrated functional similarity ofthe Notch signal pathway and presenilin function in C. elegans andhumans, it is assumed that the malfunction of the FAD presenilin mutantscan be offset in an equivalent manner by mutating a gene which ishomologous, or functionally equivalent, to ses-1 in humans. Within thecontext of the invention, it is expected, therefore, that a defect in ahuman ses-1 homolog, or a gene which is functionally equivalent, willlead to an increase in the expression of the presenilin gene which isnot affected by the FAD mutation.

The invention therefore also relates to the use of substances whichincrease the expression of a human presenilin for treating Alzheimer'sdisease, and to these substances themselves. In particular, thesubstances according to the invention include those which activate humanpresenilin gene 1 or presenilin gene 2 and increase the expression ofhuman presenilin protein 1 or human presenilin protein 2. The substancesaccording to the invention also include those which, in C. elegans, leadto a change in ses-1 activity and/or increase sel-12 or hop-1 presenilinactivity. The invention also encompasses pharmaceutical compositionswhich comprise these substances.

In an analogous manner, this invention can also be of benefit in thetreatment of the sporadic cases of Alzheimer's disease which areage-dependent and not coupled to mutations. In this connection, it isexpected that the decrease in expression will, by increasing presenilinactivity, decrease the formation of Aβ4, in particular of Aβ42, andthereby chronologically delay the onset of the disease.

BRIEF DESCRIPTION OF THE FIGURES AND OF THE SEQUENCE LISTING

FIG. 1: Ses-1 mutations

FIG. 2: Northern blot, hybridized with a hop-1-specific probe

FIG. 3: 5′ end of the ses-1 mRNAs which were found

FIG. 4: Domain structure prediction for SES-1 protein

SEQ ID NO: 1: SES-1 amino acid sequence, long form

SEQ ID NO: 2: SES-1 amino acid sequence, short form

SEQ ID NO: 3: cDNA which encodes the long form of the SES-1 protein,from ATG to stop (yk64e9)

SEQ ID NO: 4: cDNA which encodes the short form of the SES-1 protein,from ATG to stop (yk247e5)

SEQ ID NO: 5: cDNA which encodes the long form of the SES-1 protein,from ATG to stop (yk64e9)+noncoding 3′ end region

SEQ ID NO: 6: cDNA which encodes the long form of the SES-1 protein,from ATG to stop (yk64e9)+noncoding 3′ end region

SEQ ID NO: 7: Sequence of pBy1015; this segment complements the defectof the ses-1 null mutant

The invention is explained in more detail below with the aid ofexamples.

Experimental Section

Isolating ses-1 mutants

EMS and UV/TMP mutagenesis were carried out in accordance with publishedmethods (elegans.swmed.edu). In order to identify sel-12 suppressors, L4stage sel-12 mutants were exposed to the mutagen (e.g. ethylmethanesulfonate) and, after a reconvalescence period, were plated outin groups of 4 or 5 on 9 cm Petri dishes. In all the tests, the egglaying of the animals was analyzed in the F2 stage after themutagenesis. Sel-12 null mutants did not normally lay any eggs. The F3generation of the isolated animals was tested once again and thoseanimals which laid at least 5 eggs per adult animal in both generationswere isolated. In this way, homozygous suppressor strains weregenerated. All the mutations were backcrossed several times withwild-type animals in order to remove background mutation. Powerfulsuppressor mutations led to the egg-laying behavior of the animals beingcomparable to that of the wild-type animals despite the sel-12 nullmutant being homozygous. One such suppressor mutation was termed anses-1 mutation. Typically, ses-1/ses-12 double mutants laid approx.250-350 eggs per animal.

The ses-1 Phenotype

Mutations in ses-1 suppress the egg-laying defect of sel-12 mutantscompletely. Under the stereomicroscope, it is possible to distinguish 3aspects of the sel-12 egg-laying defect. About 75% of all thesel-12(ar171) animals exhibit a marked bulging of the vulva, which isknown as the protruding vulva phenotype (Pvl). The precise reason forthis defect has not been reported. Virtually all the sel-12(ar171)animals contain more eggs in the uterus than do comparable wild-typeanimals, whose constant egg number is approx. 15 whereas this number isup to 60 eggs in the case of sel-12. This leads to the animals becomingdistended, i.e. what is described as the Egl phenotype. The eggsaccumulate in the uterus and the embryos hatch within the uterus andcontinue to develop in the mother, which in virtually 100% of all casesdies prematurely in connection with this. The phenotype is termed the“bag of worms” phenotype. Wild-type animals are observed extremelyrarely in homozygous sel-12 culture. Ses-1 mutations suppress all threeaspects of this sel-12(ar171) phenotype virtually completely.Sel-12(ar171); ses-1 animals are slightly smaller than wild-type animalsand contain fewer eggs in the uterus than normal (a weakEgl-constitutive phenotype). This suggests that egg laying in the doublemutant is slightly overstimulated. Ses-1 allelic animals also suppressthe egg-laying defects of all the other sel-12 mutations which have beendescribed. This proves that ses-1 mutations do not act in anallele-specific manner.

Cloning the ses-1 gene locus

Ses-1 was mapped on the left arm of chromosome X. For this,sel-12(ar171) ses-1 strains were positioned using a genetic markermutation, egg-laying-defective (Egl) recombinant animals were isolatedand a test was carried out to determine how many of these animals hadreceived the marker mutation. In order to map ses-1 more closely, thepresence/absence of ses-1 mutations in a strain was measured bysuppression of the sel-12(ar171) egg-laying defect. For this,sel-12(ar171) ses-1 males were crossed with AB double-mutant strains,with A and B being two mutations which are located to the right ofsel-12 on the genetic map and with B being the right-hand of the twomarkers. Recombinants which were only B recombinants, and which did notcarry the A mutation, were then sought. As a result of thisrecombination method, all the recombinants should contain sel-12(ar171)apart from those which carried a double recombination. By means of aseries of crossing experiments, ses-1 was mapped between dpy-23(e840)and lin-2(e678), approx. ⅕th of the distance from dpy-23(e840) to lon-2.All the experiments indicated that ses-1 mutations bring about a loss offunction. In order to demonstrate this, ses-1 mutations were positionedby way of two free duplications. Although the ses-1 alleles by108 andby109 mutations do not complement each other, the possibility existedthat both represent mutations in two different genes on the left arm ofchromosome X. For this reason, by108 and by109 were initially mappedindependently of each other. In order to verify the mappings, twoduplications, i.e. mnDp31 and mnDp32, were transferred into the strainsel-12(ar171) ses-1(by108) lon-2(e678) and tested for egg laying.

The strain sel-12(ar171) ses-1(by108) lon-2(e678); nDp31, which carriestwo mutated copies of the ses-1 gene on the X chromosome, and alsopossesses a wild-type copy on an extrachromosomal array DNA, possessesan entirely ses-1 wild-type phenotype, which proves that the testedmutants are function-loss mutants. By linearly interpolating the data,ses-1 was located approx. 50 kb to the left of dpy-23(e840) and thetransgenic suppression was subsequently tested using cosmids of thischromosome region.

Transgene Rescue of the ses-1 Defect

Transgenes were injected into the strain sel-12(ar171) ses-1(by108) andthe antisuppressor activity of the transfected clones was tested. Allthe clones were injected at a concentration of 20 ng/μl together with100 ng of pRF4/μl and 20 ng of pBY218/μl as transformation markers. Theses-1 phenotype was completely offset (rescued) by injecting the cosmidF46H6. After that, F46H6 subclones, purified restriction fragments andPCR products from this cosmid were tested in order to elucidate theidentity of the ses-1 gene.

Isolating F46H6 subfragments

F46H6 was cut with various restriction enzymes. Subfragments werepurified using Qiaquick gel extraction kits. A 21.5 kb Ncol fragment anda 0.2 kb BamHI fragment (in the plasmid pBY1015) were injected directlyand (both) rescue the phenotype. The minimum rescuing fragment wasreduced to a sequence of 4 kb. This segment contains only one gene,which encodes a protein possessing a large number of zinc fingers.

Structure of ses-1

Two cDNAs of ses-1 were identified and contained 1.9 kb and 2.1 kb ofsequence. The two cDNAs contain identical DNA sequences and exon-intronboundaries, with one exception: the larger clone carries an additionalintron while the small clone has a 5′ end which is longer by 6 bases (inthis regard, see FIG. 3).

The sequenced cDNAs differ at several locations from the gene predictedin the databases. In the first place, the 5′ end which was found ismarkedly shorter than predicted by computer analysis. In the secondplace, sequence differences were found between both cDNAs and thegenomic sequences, published on the Internet, of the region on cosmidsF46H6 and C07A12 (neighbor cosmid) (FIG. 3).

In addition, ses-1 is trans-spliced to an SL1 leader sequence. TheSL1-specific product was sequenced. It was found that the leadersequence is connected immediately 5′ to the two predicted 5′ ends of thecDNAs.

In Northern blot experiments, a band which can be detected in all the C.elegans development stages was only found in the case of the longercDNA. The shorter cDNA could be an artifact; alternatively, it mightonly appear in small, undetectable quantities but could neverthelessperfectly well exhibit biological activity.

Intracellular Location

A ses-1 fusion gene was expressed in a bacculovirus SF9 cell system andthe location was identified by immunostaining. Ses-1 is located in thenucleus; this is in agreement with its predicted role as a transcriptionfactor.

Expression

The Northern blot analysis showed that ses-1 transcripts are expressedin all development stages (FIG. 2).

The ses-1 Null Phenotvpe

By131 is a mutant which can no longer have any ses-1 function (deletionof the entire gene). However, this deletion also eliminates neighboringgenes. By135 leads to a reading frame shift, as described, and shouldtherefore be the most powerful ses-1 mutant. Ses-1(by135) and by131animals were investigated. Both strains of phenotypically wild-type,both morphologically and on the basis of their egg-laying behavior. Theprogeny numbers of the two animals are somewhat lower than in the wildtype. Both alleles completely suppress the sel-12 phenotype of allalleles and also lead to the sel-12 alleles having wild-type progenynumber. It can therefore be concluded that ses-1 is a specificsuppressor of sel-12.

All the defects of sel-12 mutants, or of sel-12, hop-1 double mutants,can be atributed to effects of these presenilins in the lin-12 and glp-1Notch signaling pathway.

Since ses-1 is a nuclearly expressed gene, which could act as atranscription factor, an investigation was carried out to determinewhether the ses-1 mutations are able to alter the transcription of othergenes of the lin-12/glp-1 Notch signaling pathway. Stage-specific RNAswere isolated from the wild type and various ses-1 mutant strains andanalyzed in Northern blots. The samples were tested by hybridizing withhop-1 cDNAs. Hop-1 is strongly expressed in the eggs of wild-typeanimals but is almost impossible to detect in the L1 larval stage.Expression increases slowly from the L2 stage to the adult stage andreaches its peak in adult animals. In sel-12(ar171) animals, theexpression of hop-1 was to a large extent identical. In sel-12(ar171)ses-1(by135) animals and ses-1(by135) animals, expression differedsignificantly in one stage: it was clearly possible to detect hop-1 inthe L1 stage. The expression of hop-1 in the L1 stage was alsosignificantly greater in ses-1 alleles by108 and by136. This means thatses-1 mutants achieve suppression of sel-12 defects by markedlyderepressing the expression of hop-1, and HOP-1 protein is presumablyable to assume the function of the defective SEL-12.

1-32. (Cancelled).
 33. An isolated nucleic acid molecule that encodesSES-1 or a mutated form of SES-1.
 34. An isolated nucleic acid asdescribed in claim 33, wherein the nucleic acid is a DNA with one ormore alterations at one or more of positions 1393, 1784, 1481, 3052,6698, 549, 25042, 27543, and 1163 relative to a starting ATG.
 35. Anisolated nucleic acid as described in claim 34, wherein the one or morealterations is selected from the group consisting of a C to T conversionat position 1393, a G to A conversion at position 1784, a CAA to AACconversion at position 1481, a deletion from position 3052 towardsposition 6698 by a single A base pair insertion, an A removal atposition 549, a deletion from the EcoR1 side at position 25042 of F4686to the Scal side at position 27543, and a T to A conversion at position1163.
 36. An isolated nucleic acid molecule as described in claim 33,which is a DNA molecule, a cDNA molecule, a genomic DNA molecule, or anRNA molecule.
 37. An isolated nucleic acid molecule as described inclaim 33, wherein the SES-1 has an amino acid sequence identical to, oressentially the same as at least one of SEQ ID NO: 1 and SEQ ID NO:2.38. An isolated nucleic acid molecule as described in claim 33,comprising a nucleotide sequence selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, orcomprising a sequence that is complementary thereto.
 39. An isolatednucleic acid molecule as described in claim 33, comprising a nucleotidesequence having at least 75% sequence similarity to a nucleotidesequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, or a sequence complementary thereto.40. An isolated nucleic acid molecule that hybridizes with an isolatednucleic acid molecule as described in claim 33 under conditions of0.1×SSC at 68 degrees centigrade.
 41. A vector that comprises anisolated nucleic acid molecule or a fragment thereof as described inclaim
 33. 42. A vector as described in claim 41, which comprises apromoter for expression in a cell of the nematode C. elegans.
 43. Avector as described in claim 41, which is in the form of a plasmid, acosmid, a bacmid, a virus or a bacteriophage.
 44. An SES-1 proteincomprising the amino acid sequence of at least one of SEQ ID NO: 1 andSEQ ID NO: 2, a mutated version thereof, or a fragment thereof.
 45. AnSES-1 protein as described in claim 44, comprising one or moreadditions, deletions, or alterations at one or more amino acid positionscorresponding to one or more amino acids of at least one of SEQ ID NO: 1and SEQ ID NO:
 2. 46. A mutated protein as described in claim 45,encoded by a nucleic acid molecule comprising one or more mutations atnucleic acid positions 1393, 1784, 1481, 3052, 6698, 549, 25042, 27543,and 1163 relative to a starting ATG.
 47. An antibody having bindingactivity against an SES-1 protein as described in claim
 44. 48. Anantibody as described in claim 46, comprising a monoclonal antibody or apolyclonal antibody.
 49. A transgenic, nonhuman organism or cell thatcomprises a foreign nucleic acid molecule having a sequence as describedin claim
 39. 50. A transgenic organism as described in claim 49, whereinthe organism is C. elegans.
 51. A transgenic C. elegans organism asdescribed in claim 50, which expresses an SES-1 allele that decreases,amplifies or eliminates the activity of SES-1.
 52. A transgenic C.elegans organism as described in claim 51, which expresses an SES-1allele that offsets the egg-laying defect phenotype (Egl) in sel-12 C.elegans mutants.
 53. A transgenic C. elegans organism as described inclaim 51, which expresses an SES-1 allele that increases the activity ofhop-1 or sel-12.
 54. A method of producing a pharmaceutical for treatingneurodegenerative disease, comprising expressing an isolated nucleicacid molecule as described in claim 33, or a functionally similar gene,to produce a gene product, and isolating the gene product.
 55. A methodfor investigating neurodegenerative disease, comprising generating atransgenic organism by incorporating a nucleic acid molecule asdescribed in claim 33 into the organism, such that the introducednucleic acid functions to affect the activity of SES-1.
 56. A method forinvestigating neurodegenerative disease, comprising monitoring SES-1activity in the presence of a test substance, and identifying orcharacterizing an effect of the substance on the monitored activity. 57.The method of claim 56, wherein the SES-1 activity is monitored in atransgenic organism or cell.
 58. The method of claim 57, wherein thetransgenic organism is C. elegans.
 59. The method of claim 57, wherein adecrease or elimination of SES-1 activity is detected and correlatedwith the presence or abundance of the test substance.
 60. The method ofclaim 57, wherein an increase in the activity of hop-1 or sel-12 ismonitored and correlated with the presence or abundance of the testsubstance.
 61. A method of identifying a compound for treating orpreventing Alzheimer's disease, comprising providing a transgenic C.elegans organism as described in claim 50, exposing the organism to thecompound, and detecting an effect of that exposure.
 62. A method fordiscovering a medicament for treating Alzheimer's disease, comprisingobtaining a substance identified by the method of claim 50, anddetermining the effect of that substance on the expression of presenilin1 or presenilin
 2. 63. A substance that increases the expression ofhuman presenilin 1 or presenilin 2, comprising a molecule discovered bythe method of claim
 62. 64. A pharmaceutical composition that comprisesa substance as described in claim 63.